Evidence for Proline Catabolic Enzymes in the Metabolism of Thiazolidine Carboxylates.

Thiazolidine carboxylates such as thiazolidine-4-carboxylate (T4C) and thiazolidine-2-carboxylate (T2C) are naturally occurring sulfur analogues of proline. These compounds have been observed to have both beneficial and toxic effects in cells. Given that proline dehydrogenase has been proposed to be a key enzyme in the oxidative metabolism of thioprolines, we characterized T4C and T2C as substrates of proline catabolic enzymes using proline utilization A (PutA), which is a bifunctional enzyme with proline dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH) activities. PutA is shown here to catalyze the FAD-dependent PRODH oxidation of both T4C and T2C with catalytic efficiencies significantly higher than with proline. Stopped-flow experiments also demonstrate that l-T4C and l-T2C reduce PutA-bound FAD at rates faster than proline. Unlike proline, however, oxidation of T4C and T2C does not generate a substrate for NAD+-dependent GSALDH. Instead, PutA/PRODH oxidation of T4C leads to cysteine formation, whereas oxidation of T2C generates an apparently stable Δ4-thiazoline-2-carboxylate species. Our results provide new insights into the metabolism of T2C and T4C.

The SUN1-SPDYA interaction plays an essential role in meiosis prophase I.

Chromosomes pair and synapse with their homologous partners to segregate correctly at the first meiotic division. Association of telomeres with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex composed of SUN1 and KASH5 enables telomere-led chromosome movements and telomere bouquet formation, facilitating precise pairwise alignment of homologs. Here, we identify a direct interaction between SUN1 and Speedy A (SPDYA) and determine the crystal structure of human SUN1-SPDYA-CDK2 ternary complex. Analysis of meiosis prophase I process in SPDYA-binding-deficient SUN1 mutant mice reveals that the SUN1-SPDYA interaction is required for the telomere-LINC complex connection and the assembly of a ring-shaped telomere supramolecular architecture at the nuclear envelope, which is critical for efficient homologous pairing and synapsis. Overall, our results provide structural insights into meiotic telomere structure that is essential for meiotic prophase I progression.

In silico analysis and prediction of transcription factors of the proteins interacting with astrocyte elevated gene-1.

Multifunctional in nature, the protein Astrocyte Elevated Gene-1 (AEG-1) controls several cancers through protein-protein interactions. Although, specific physiological processes and molecular functions linked with AEG-1 interactors remain unclear. In our present study, we procured the data of AEG-1 interacting proteins and evaluated their biological functions, associated pathways, and interaction networks using bioinformatic tools. A total of 112 proteins experimentally detected to interact with AEG-1 were collected from various public databases. DAVID 6.8 Online tool was utilized to identify the molecular functions, biological processes, cellular components that aid in understanding the physiological function of AEG-1 and its interactors in several cell types. With the help of integrated network analysis of AEG-1 interactors using Cytoscape 3.8.0 software, cross-talk between various proteins, and associated pathways were revealed. Additionally, the Enrichr online tool was used for performing enrichment of transcription factors of AEG-1 interactors' which further revealed a closely associated self-regulated interaction network of a variety of transcription factors that shape the expression of AEG-1 interacting proteins. As a whole, the study calls for better understanding and elucidation of the pathways and biological roles of both AEG-1 and its interactor proteins that might enable their application as biomarkers and therapeutic targets in various diseases in the very near future.

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast.

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27-760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 µM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

Deafness mutation D572N of TMC1 destabilizes TMC1 expression by disrupting LHFPL5 binding.

Transmembrane channel-like protein 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) are recognized as two critical components of the mechanotransduction complex in inner-ear hair cells. However, the physical and functional interactions of TMC1 and LHFPL5 remain largely unexplored. We examined the interaction between TMC1 and LHFPL5 by using multiple approaches, including our recently developed ultrasensitive microbead-based single-molecule pulldown (SiMPull) assay. We demonstrate that LHFPL5 physically interacts with and stabilizes TMC1 in both heterologous expression systems and in the soma and hair bundle of hair cells. Moreover, the semidominant deafness mutation D572N in human TMC1 (D569N in mouse TMC1) severely disrupted LHFPL5 binding and destabilized TMC1 expression. Thus, our findings reveal previously unrecognized physical and functional interactions of TMC1 and LHFPL5 and provide insights into the molecular mechanism by which the D572N mutation causes deafness. Notably, these findings identify a missing link in the currently known physical organization of the mechanotransduction macromolecular complex. Furthermore, this study has demonstrated the power of the microbead-based SiMPull assay for biochemical investigation of rare cells such as hair cells.

[Establishment of fluorescence immunochromatography detection for cytoskeleton-associated protein 4].

A rapid and simple method to detect tumor markers in liver cancer was established by combining immunochromatography technique with fluorescent microsphere labeling. According to the principle of double antibody sandwich, the cytoskeleton-associated protein 4 (CKAP4) paired antibody was used as the labeled and coated antibody, and the goat anti-rabbit polyclonal antibody was used as the quality control line coated antibody in the preparation of the CKAP4 fluorescent immunochromatographic test strips. After the preparation, the test strips were evaluated on various performance indicators, such as linearity, precision and stability. The CKAP4 immunochromatographic strip prepared by time-resolved fluorescent microspheres had high sensitivity, and good specificity. Its precision was within 15%, recovery between 85% and 115%, and linear range between 25 and 1 000 pg/mL. The test strip could be kept stable at 37 °C for 20 days, and it correlated well with commercial ELISA kits. The CKAP4 fluorescence immunochromatography method can quantitatively detect the content of CKAP4 in serum. Furthermore, it is rapid, sensitive, simple, economical and single-person operation. This method has the potential of becoming a new method for the diagnosis and treatment of liver cancer.

Membrane Protein Solubilization and Quality Control: An Example of a Primary Active Transporter.

When purifying a membrane protein, finding a detergent for solubilization is one of the first steps to master. Ideally, only little time is invested to identify the best-suited detergent, which on the one hand would solubilize large amounts of the target protein but on the other hand would sustain the protein's activity. Here we describe the solubilization screen and subsequent activity assay we have optimized for the bacterial P-type ATPase KdpFABC. In just 2 days, more than 70 detergents were tested for their solubilization potential. Afterwards, a smaller selection of the successful detergents was assayed for their ability to retain the activity of the membrane protein complex.

A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2.

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

Human Amnion Membrane Proteins Prevent Doxorubicin-Induced Oxidative Stress Injury and Apoptosis in Rat H9c2 Cardiomyocytes.

Doxorubicin (DOX) is widely used as an effective chemotherapy agent in cancer treatment. Cardiac toxicity in cancer treatment with DOX demand urgent attention and no effective treatment has been established for DOX-induced cardiomyopathy. It has been well documented that human amniotic membrane proteins (AMPs), extracted from amnion membrane (AM), have antioxidant, anti-apoptotic, and cytoprotective properties. Therefore, in this study, we aimed to investigate the protective effects of AMPs against cardiotoxicity induced by DOX in cultured rat cardiomyocyte cells (H9c2). DOX-induced cell injury was evaluated using multi-parametric assay including thiazolyl blue tetrazolium bromide (MTT), the release of lactic dehydrogenase (LDH), intracellular Ca2+ , reactive oxygen species (ROS) levels, cellular antioxidant status, mitochondrial membrane potential (ΔΨm), malondialdehyde (MDA), and NF-κB p65 DNA-binding activity. Moreover, expression profiling of apoptosis-related genes (P53, Bcl-2, and Bax) and Annexin V by flow cytometry were used for cell apoptosis detection. It was shown that AMPs pretreatment inhibited the cell toxicity induced by DOX. AMPs effectively attenuated the increased levels of LDH, Ca2+ , ROS, and MDA and also simultaneously elevated the ΔΨm and antioxidant status such as superoxide dismutase (SOD) and Catalase (CAT) in pretreated H9c2 cardiomyocytes. Besides, the activity of NF-kB p65 was reduced and the p53 and Bax protein levels were inhibited in these myocardial cells subjected to DOX. These findings provide the first evidence that AMPs potently suppressed DOX-induced toxicity in cardiomyocytes through inhibition of oxidative stress and apoptosis. Thus, AMPs can be a potential therapeutic agent against DOX cardiotoxicity.

Lactobionamide-based fluorinated detergent for functional and structural stabilization of membrane proteins.

Membrane proteins (MPs) are important drug discovery targets for a wide range of diseases. Conventional detergents such as n-Dodecyl ß-D-maltoside have been used largely and efficiently to solubilize MPs with varying degrees of success concerning MPs functionality and stability. Fluorinated surfactants (FSs) have shown a great potential for the stabilization of various MPs. However, so far only a limited number of reports have demonstrated the ability of FSs to solubilize MPs from biological membranes. We report herein the use of a fluorinated lactobionamide-based detergent named FLAC6 for functional and structural stabilization of membrane proteins. We first demonstrated that FLAC6 efficiently solubilized three membrane proteins i.e. the native adenosine receptor A2AR, a G protein-coupled receptor, and two native transporters AcrB and BmrA. The resulting affinity purified MPs were highly pure, homogenous and aggregates free. Furthermore, the functionality of each MP was well maintained. Finally, striking overstabilization features were observed. Indeed, the Tm of native A2AR, AcrB and BmrA could be improved by 7, ~9 and ~ 23 °C, respectively when FLAC6 was used instead of the reference detergent. This work illustrates that FLAC6 is an efficient tool to maintain structural and functional integrities of different MPs belonging to different classes, providing a new avenue for functional stabilization of highly druggable and challenging membrane proteins involved in unmet medical needs.

Purification and mutagenesis studies of TANC1 ankyrin repeats domain provide clues to understand mis-sense variants from diseases.

TANC1 and its close relative TANC2 are two important synaptic scaffold proteins which play critical roles in regulating densities of synaptic spines and excitatory synapse strength. Recent studies indicated TANC1 and TANC2 are candidate genes of several neurodevelopmental disorders (NDD). So far, the biochemical properties of TANC1/2 proteins remain largely unknown. In this study, Ankyrin-repeats (AR) domain of TANC1 was expressed and purified using Escherichia coli. (E. coli.) cells, which showed low solubility and stability after removing the maltose binding protein (MBP) tag. Sequence analysis revealed that the TANC1 AR domain is lack of canonical N, C-capping units. By introducing two point mutations in the C-capping unit and replacing the N-capping unit, monomeric and well-folded TANC1 AR domain was purified and characterized by size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) and circular dichroism spectroscopy (CD). In addition, mutations from intellectual disability (ID) patients and cancer patients were imported into the TANC1 AR domain. The ID mutant exhibited marginal effects in terms of conformation and protein folding stability changes. By contrast, the cancer mutants dramatically decreased protein solubility. Combined with structural prediction, we speculated that mis-sense variants tested in this study may either affect protein folding or disrupt the interaction between TANC1/2 AR domains and their binding partners.

Hydrogenated Diglucose Detergents for Membrane-Protein Extraction and Stabilization.

We report herein the design and synthesis of a novel series of alkyl glycoside detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane-, and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents, respectively. Micellization in aqueous solution was studied by isothermal titration calorimetry, 1H NMR spectroscopy, and surface tensiometry. Critical micellar concentration values were found to decrease by a factor of ∼10 for each pair of methylene groups added to the alkyl chain, ranging from ∼0.05 to 9 mM for DDDG and ODG, respectively. Dynamic light scattering and analytical ultracentrifugation sedimentation velocity experiments were used to investigate the size and composition of the micellar aggregates, showing that the aggregation number significantly increased from ∼40 for ODG to ∼80 for DDDG. All new compounds were able to solubilize membrane proteins (MPs) from bacterial membranes, insect cells, as well as the Madin-Darby canine kidney cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter (BmrA) were solubilized efficiently. Striking thermostability improvements of +13 and +8 °C were observed when ODG and DDG were, respectively, applied to wild-type and full-length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of membrane proteins (MPs) and thus makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging MP targets.

Development of a multiplexed giant magnetoresistive biosensor array prototype to quantify ovarian cancer biomarkers.

In this work, we developed benchtop and handheld Giant Magnetoresistive (GMR) biosensing systems that serve as platforms for detecting a wide variety of protein biomarkers for human diseases. System development included spintronic and nanomagnetic materials, biomolecular chemistry, electronic circuitry, analog and digital signal processing, firmware programming, user interface programming on both PC and Android smartphone, communications over both USB and Bluetooth, and mechanical integration. In this work, we demonstrated the benchtop GMR biosensing system in the context of ovarian cancer assay development. The prototype system delivered the required performance in terms of high-sensitivity multiplex assays in a portable format with enough flexibility to serve as a platform for ovarian cancer and many other diseases. We achieved multiplex detection of cancer antigen 125 (CA125 II), human epididymis protein 4 (HE4), and interleukin 6 (IL6), with limits of detection (LOD) as low as 3.7 U/mL, 7.4 pg/mL, and 7.4 pg/mL, respectively.

Lysis Buffer Choices Are Key Considerations to Ensure Effective Sample Solubilization for Protein Electrophoresis.

The efficient extraction of proteins of interest from cells and tissues can be challenging. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 and the transcriptional repressor Snail from choriocarcinoma cells using NP-40 and RIPA lysis buffer. We also show the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness with the often utilized RIPA lysis buffer for solubilization of heat shock proteins (HSP) B1 and B5 and the cytoplasmic adapter protein integrin-linked kinase (ILK) from smooth muscle. Overall, the results demonstrate the importance of optimizing lysis buffers for specific protein solubilization prior to finalizing the experimental workflow.

Molecular Classification of Lymph Node Metastases Subtypes Predict for Survival in Head and Neck Cancer.

PURPOSE: In advanced stage head and neck squamous cell cancers (HNSCC), approximately half of the patients with lymph node metastases (LNM) are not cured. Given the heterogeneous outcomes in these patients, we profiled the expression patterns of LNMs to identify the biological factors associated with patient outcomes.Experimental Design: We performed mRNAseq and miRNAseq on 72 LNMs and 29 matched primary tumors from 34 patients with HNSCC. Clustering identified molecular subtypes in LNMs and in primary tumors. Prediction Analysis of Microarrays algorithm identified a 73-gene classifier that distinguished LNM subtypes. Gene-set enrichment analysis identified pathways upregulated in LNM subtypes. RESULTS: Integrative clustering identified three distinct LNM subtypes: (i) an immune subtype (Group 1), (ii) an invasive subtype (Group 2), and (iii) a metabolic/proliferative subtype (Group 3). Group 2 subtype was associated with significantly worse locoregional control and survival. LNM-specific subtypes were not observed in matched primary tumor specimens. In HNSCCs, breast cancers, and melanomas, a 73-gene classifier identified similar Group 2 LNM subtypes that were associated with worse disease control and survival only when applied to lymph node sites, but not when applied to other primary tumors or metastatic sites. Similarly, previously proposed prognostic classifiers better distinguished patients with worse outcomes when applied to the transcriptional profiles of LNMs, but not the profiles of primary tumors. CONCLUSIONS: The transcriptional profiles of LNMs better predict outcomes than transcriptional profiles of primary tumors. The LNMs display site-specific subtypes associated with worse disease control and survival across multiple cancer types.

Identification of midgut membrane proteins from different instars of Helicoverpa armigera (Lepidoptera: Noctuidae) that bind to Cry1Ac toxin.

Helicoverpa armigera is a polyphagous pest sensitive to Cry1Ac protein from Bacillus thuringiensis (Bt). The susceptibility of the different larval instars of H. armigera to Cry1Ac protoxin showed a significant 45-fold reduction in late instars compared to early instars. A possible hypothesis is that gut surface proteins that bind to Cry1Ac differ in both instars, although higher Cry toxin degradation in late instars could also explain the observed differences in susceptibility. Here we compared the Cry1Ac-binding proteins from second and fifth instars by pull-down assays and liquid chromatography coupled to mass spectrometry analysis (LC-MS/MS). The data show differential protein interaction patterns of Cry1Ac in the two instars analyzed. Alkaline phosphatase, and other membrane proteins, such as prohibitin and an anion selective channel protein were identified only in the second instar, suggesting that these proteins may be involved in the higher toxicity of Cry1Ac in early instars of H. armigera. Eleven Cry1Ac binindg proteins were identified exclusively in late instar larvae, like different proteases such as trypsin-like protease, azurocidin-like proteinase, and carboxypeptidase. Different aminopeptidase N isofroms were identified in both instar larvae. We compared the Cry1Ac protoxin degradation using midgut juice from late and early instars, showing that the midgut juice from late instars is more efficient to degrade Cry1Ac protoxin than that of early instars, suggesting that increased proteolytic activity on the toxin could also explain the low Cry1Ac toxicity in late instars.

Purification and Functional Characterization of the C-Terminal Domain of the ß-Actin-Binding Protein AIM1 In Vitro.

The protein absent in melanoma 1 (AIM1) is a member of the ßγ-crystal lens superfamily that is associated with the development of multiple cancers. The binding of AIM1 to ß-actin affects the migration and invasion of prostate cancer epithelial cells. The C-terminus of AIM1 is required for the ß-actin interaction. However, the characteristics of AIM1 in vitro and the interaction mode between AIM1 and ß-actin remain unknown. We describe novel methods to prepare pure recombinant AIM1 and identify possible binding modes between AIM1 and ß-actin; we also obtain the crystal of the first two ßγ-crystallin domains of AIM1 (g1g2) for future structural biology research. We first express and purify AIM1 after cloning the sequence into a modified pET-28a_psp expression vector. Next, we define the minimum unit formed by the ßγ-crystallin domain repeats that bound to ß-actin and perform its physiological function. Finally, we made the structural model of the AIM1 g1g2 that can be used to guide future biomedical investigations and prostate cancer research.

Cytosolic and Transmembrane Protein Extraction Methods of Breast and Ovarian Cancer Cells: A Comparative Study.

Efficient extraction of proteins is a great challenge for numerous downstream proteomic analyses. During the protein extraction procedure, it is critical to maintain the conformational stability, integrity, as well as higher yield of the protein. To do so, 5-different lysis buffers of Tris and HEPES have been used as the primary buffering reagents with variable compositions at different concentrations and pH using human cancer cells. In this study, different protein lysates of human breast cancer cells T47D and MDA-MB-231 and ovarian cancer cell PA-1 were subjected to run SDS-PAGE for separation of proteins based on their molecular size, followed by Coomassie blue, silver staining, and immunoblot assays to compare the extraction yield of total cytoplasmic proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the integral membrane protein, integrin ß-1. Our results revealed that Tris-based lysis buffer with 50 mM concentration, pH 7.5, is relatively the efficient and reliable protein extraction method for a wide range of MW subcellular markers, cytoplasmic GAPDH and transmembrane integrin ß-1 proteins. We anticipate that this simple and cost-effective protein extraction protocol might be extremely useful across a broad range of subcellular proteins in different biologic samples.